Cluster Differentiation 90 (CD90) is a cell adhesion molecule and the smallest member of the immunoglobulin superfamily with a molecular weight of 25-35 KDa. CD90 is also known as thymocyte differentiation antigen-1 (Thy-1) and is a glycoprotein anchored to the cell surface via a glycosylphosphatidylinositol (GPI) motif. In humans, CD90 is expressed on stem cells including Mesenchymal Stem Cells (MSCs), Hematopoietic Stem Cells (HSCs) and Keratinocyte Stem Cells (KSCs) and at varying levels on non-lymphoid tissues such as fibroblasts, neurons and activated endothelial cells. Keratinocytes form the predominant cell type in the epidermis, the outermost layer of the skin, constituting 90% of the cells found there. Those keratinocytes found in the basal layer of the skin are referred to as “basal cells” or “basal keratinocytes.
CD90 expressing skin cells are functioning in inflammation and wound healing by synthesizing and releasing growth factors, cytokines and extracellular matrix components to assist in repairing damaged skin tissue. For instance, monocyte CD90 ligand binds to CD90 on activated endothelial cells during inflammation, focusing the immune cell migration to the sites of inflammation, tissue injury and infection.
Erythropoietin (EPO) is a pleiotropic cytokine glycoprotein that was initially identified as a regulator of red blood cell production in response to hypoxia. The mature human 165 amino acid-long EPO protein sequence is presented by SEQ ID NO: 1
(SEQ ID NO: 1)APPRLICDSRVLERYLLEAKEAENITTGCAEHCSLNENITVPDTKVNFYA WKRMEVGQQAVEVWQGLALLSEAVLRGQALLVNSSQPWEPLQLHVDKAVS GLRSLTTLLRALGAQKEAISPPDAASAAPLRTITADTFRKLFRVYSNFLR GKLKLYTGEACRTGDRand consists of four α-helices, forming a compact globular structure. Human recombinant erythropoietin (expressed in mammalian cells) contains three N-linked and one O-linked oligosaccharide chains which together comprise about 40% of the total molecular weight of the glycoprotein. N-linked glycosylation occurs at asparagine residues (Asn) located at positions 24, 38 and 83 whereas O-linked glycosylation occurs at a serine residue (Ser) located at position 126.
According to the classical original understanding, EPO induces haematopoiesis by dimerizing EPO receptor molecules, which leads to the activation of the EPO receptor-associated Janus tyrosine kinase 2 (Jak2) and secondary signaling molecules such as Stat5 (Brines and Cerami, Nat Rev Neurosci, 2005; 6:484-94). Recombinant EPO is widely used for the treatment of anemia of various origins.
EPO also displays tissue protection properties. In the central nervous system, EPO and EPO receptor are expressed by neurons, glial cells and cerebrovasculature endothelium. EPO was shown to be neurotrophic and neuroprotective in vitro and in animal models of neuronal injury associated with trauma, stroke, ischemia, inflammation and epileptic seizures. The beneficial effects of EPO were also demonstrated in clinical studies of stroke, schizophrenia and progressive multiple sclerosis. EPO protects neurons both directly, by preventing apoptosis, and indirectly, by modulating inflammatory processes and stimulating neurogenesis and angiogenesis (Wang et al., Stroke 2004; 35:1732-7).
It has been shown that some of the cytoprotective effects of erythropoietin are mediated through its binding to heterodimers containing the canonical erythropoietin receptor and the common beta receptor, ßcR (Brines et al., Proc Natl Acad Sci USA 2004; 101: 14 907-14 912). Interestingly, carbamylated erythropoietin binds to these heteroreceptors and exerts tissue-protective effects, whereas it does not bind to the classical erythropoietin receptor and does not stimulate erythropoiesis. ßcR is not required for erythropoiesis. It is assumed that ßcR in combination with the EpoR expressed by nonhematopoietic cells constitutes a tissue-protective receptor, thus creating a tissue-protective heteroreceptor.
A tissue regenerative and wound healing promoting effect of EPO in skin and other tissues was demonstrated numerous times in vivo and in vitro by systemic and even topical application forms (e.g.: Giri et al, Drug Design, Development and Therapy 2015:9; Günter et al., J Transplant Ste Cell Biol 2(1): 4, 2015; Hamed et al., Wound Rep Reg (2014) 22 23-33; WO 2004/001023; WO 2005/063965; WO 2009/083203).
However, with respect to the regeneration of skin defects and injuries, some problems have become evident that restrict the usability of EPO and its analogs to a direct application of such compounds to the defected or injured skin wounds since these polypeptides do not penetrate the skin. Unless applied by intravenously, subcutaneously or by intramuscular injection, the protein erythropoietin or analogous cannot enter the body.
Topical application of EPO alone to the skin will fail, because it lacks skin permeability on its own. This severely limits the applicability of the molecule. Even in these cases it is controversial whether the systemic application has a regenerative potential at all but rather supports the conventional proliferation and maturation of blood cells, and not topical intact skin or tissue regeneration. It is reported that even the subcutaneous injection of 300 Units EPO/kg body weight used were not able to induce skin regeneration.
In addition, wounds are rich in proteinases known to inactivate EPO within minutes, so EPO has a limited potential to be applied topically to enhance wound regeneration due to EPO's short half-life caused by rapid proteolytic degradation. The same can be said with EPO-fragments or variants or derivatives which known in the art. Some of these EPO derivatives or fragments or variants are known to be effective in tissue protection (see, for example, EP 2 933 264, EP 2 371 855, WO 2007/019545).
The restricted availability of these EPO derived polypeptides is a dilemma especially in a situation of skin conditions that are present in aged skin, scar rich tissue or even inflammatory conditions such as neurodermitis or eczema, where even the most superficial areas of the skin are relatively intact. Not even a cosmetic activity of EPO and its analogues would be achievable.
The aim of this invention is to establish and improve applicability of EPO and EPO fragments, analogs, mimetics, variants and derivatives, to skin cells in a close geometric distance rather than a gradient and time of impact manner.
Therefore, in order to overcome the above-specified limitations, the development of new chemical entities is needed that show tissue-protective properties and are able to efficiently target competent cells in skin tissue which trigger and carry out the physiological and biological functions of skin cells to withdraw or to attenuate the damages of the skin caused, for example, by skin aging and outer and inner influences on skin cells based on pathological events.